Project Details
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Directed protein targeting to study protein-protein interactions and the biological effects of functional protein knockouts

Subject Area Biochemistry
Term from 2003 to 2011
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 5400117
 
Final Report Year 2017

Final Report Abstract

Protein-protein interactions are key for virtually all biological systems and play important roles in numerous diseases. Consequently, protein-protein interaction surfaces are attractive yet challenging targets for disease intervention. One of the most abundant protein interaction domain is the coiledcoil motif, which consists of two or more α-helices wrapped around each other. The project focused on coiled-coil mediated protein-protein interactions involved in diseases such as cancer. We aimed at (i) understanding the molecular principles driving specificity and affinity of protein-protein interactions, and (ii) generating interfering peptides (iPEP) disrupting such protein interactions for analytical as well as medical purposes. We used rational design in combination with specifically adapted in vivo and in vitro selection systems combining competitive and negative design aspects. Next to phage display we adapted proteinfragment complementation for simultaneous selection of affinity and specificity (CANDI, competitive and negative design initiative) and we devised a hitchhiker translocation assay for selection of binary and ternary complexes. Peptides directed against the protein-protein interaction domains of the medically relevant proteins c-Jun, c-Fos, c-Myc, AF10 and the microphthalmia-associated transcription factor MITF were generated, and molecular properties of selected sequences were analyzed and compared with regard to their kinetic and thermodynamic properties. Major energetic differences (≥5.6 kcal/mol) were observed between desired and non-desired interaction stabilities for a selection implementing negative design aspects relative to a conventional selection experiment, with significantly higher stability (3.2 kcal/mol) than the wild-type interaction. In addition, we programmed a 'bZIP coiled-coil interaction prediction algorithm' (bCIPA) for predicting coiled coil-mediated proteinprotein interaction of natural proteins as well as designed inhibitors. To improve proteolytic stability, D-peptides were exploited as well. Based on a designed coiled coillike D/L-heterotetramer we used a library approach to generate D-peptide binders. Phage display selection yielded one predominant peptide that differed at 13 positions from the scaffold helix. In comparison to the designed D/L-arrangement, the selected pair showed fewer intermediate states which could largely be attributed to improved electrostatic interactions. Furthermore, the influence of the core residues was investigated. Changing the all leucine core in the original assembly to valine resulted in a peptide completely devoid of binding the D-peptide target, whereas a variant with an isoleucine core interacted with the D-peptide in a significantly more specific complex than the original design. To remotely control the activity of the inhibitor, photo-switchable chemical linkers were coupled to our selected c-Jun-targeting iPEP. Light-induced switching of the linker caused folding or unfolding of the peptides rendering them active (folded) or inactive (unfolded). Switching was reversible and could be repeated in both directions. Importantly, light-activated peptides exhibited much stronger inhibition of Jun:Jun:DNA and Jun:Fos:DNA complexes and interference with gene transcription than their non-activated counter parts. The effect of the iPEPs to interfere with endogenous activator protein 1 (AP-1) activity was tested in reporter gene assays directly or indirectly dependent on AP-1. In all cases, the iPEP was able to significantly lower AP-1 dependent gene expression. In summary, tailored semirational library design paired with efficient selection techniques allowed sampling of a large sequence space to generate iPEPs that specifically interfere with their target sequences and characterization of resulting peptide pairs provided insight into molecular determinants driving affinity and specificity. Furthermore, iPEPs interfering with medically relevant proteins might become important diagnostics and therapeutics.

Publications

  • (2004). Coiled coil domains: stability, specificity, and biological implications. ChemBioChem 5(2); 170-6
    Mason, J.M. & Arndt, K.M.
  • (2006). Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design. Proc. Natl. Acad. Sci. USA 103(24), 8989-8994
    Mason, J.M., Schmitz, M.A., Müller, K.M. & Arndt, K.M.
  • (2007). Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif: A Stopped-flow Circular Dichroism Kinetic Analysis. J. Biol. Chem. 282(32), 23015-23024
    Mason, J.M., Hagemann, U.B. & Arndt, K.M.
  • (2007). Positive Aspects of Negative Design: Simultaneous Selection of Specificity and Interaction Stability. Biochemistry 46(16); 4804-14
    Mason, J.M., Müller, K.M. & Arndt, K.M.
  • (2008). Selectional and mutational scope of peptides sequestering the c-Fos coiled coil domain. J. Mol. Biol. 381(1), 73-88
    Hagemann, U.B., Mason, J.M., Müller, K.M. & Arndt, K.M.
  • (2009). Improving the Interaction of Myc- Interfering Peptides with Myc Using Molecular Dynamics Simulations. J. Pept. Sci. 15(1), 5-15
    Jouaux, E.M., Timm, B.B., Arndt, K.M. & Exner, T.E.
  • (2009). Role of Hydrophobic and Electrostatic Interactions in Coiled Coil Stability and Specificity. Biochemistry 48(43), 10380-10388
    Mason, J.M., Hagemann, U.B. & Arndt, K.M.
  • (2010). Photo-control of Coiled-coil Proteins in Living Cells. Angew. Chem. Int. Ed. 49(23), 3943-3946
    Zhang, F., Timm, K.A., Arndt, K.M. & Woolley, G.A.
  • (2014). Analysis of Selected and Designed Chimeric D- and L-α-Helix Assemblies. Biomacromolecules 15, 3296-3305
    Kükenshöner, T., Hagemann, U.B., Wohlwend, D., Räuber, C. Baumann, T. Keller S. Einsle, O., Müller K.M. & Arndt, K.M.
    (See online at https://doi.org/10.1021/bm5006883)
  • (2014). Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system. J. Struct. Biol. 186, 335–48
    Kükenshöner, T., Wohlwend, D., Niemöller, C., Dondapati, P., Speck, J., Adeniran, A.V., Nieth, A., Gerhardt, S., Einsle, O., Müller, K.M. & Arndt, K.M.
    (See online at https://doi.org/10.1016/j.jsb.2014.03.002)
 
 

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